The creation of a recombinant host suitable for high-level production of a specific product typically requires significant metabolic engineering to the native host machinery and biosynthetic pathways. In response to needs for high-level gene expression, many specialized expression vectors have been created by manipulating a number of different genetic elements that control aspects of transcription, translation, protein stability, oxygen limitation, and secretion from the host cell. More specifically, the molecular features that must be considered when designing systems for optimal gene expression include: 1.) the nature of the relevant transcriptional promoter and terminator sequences; 2.) the strength of the ribosome binding site; 3.) the final cellular location of the synthesized foreign protein; 4.) the efficiency of translation in the host organism; 5.) the intrinsic stability of the cloned gene protein within the host cell; 6.) the codon usage within the cloned gene, such that its frequency approaches the frequency of preferred codon usage of the host cell; and 7.) the number of copies of the cloned gene and whether the gene is plasmid-borne or integrated into the genome of the host cell.
Although each of the modifications described above has tremendous utility, the ease of testing each modification in a particular host organism for production of a specific protein product as a means to increase gene expression varies widely. For example, modification of the codon usage of a particular cloned gene is a tedious process, requiring nucleotide base pair modifications throughout the gene of interest. In contrast, one of the easiest means to increase gene expression involves increasing the number of copies of the plasmid-borne cloned gene(s), either by: 1.) increasing the number of copies of the cloned gene within each expression plasmid; or 2.) increasing the copy number of the plasmid on which the gene to be expressed resides. The former requires additional cloning for each gene that is to be expressed within a particular expression plasmid and multiple copies of an identical gene may be unstable due to homologous recombination. The latter method requires synthesis of a suite of modified expression plasmids having altered plasmid copy number suitable for the particular host organism of interest; however, numerous different cloned genes can readily be tested within this suite of plasmids to determine the optimal ratio of gene copy number to gene expression level.
Concerning plasmid copy number; it is known that plasmids must control their replication so that the copy number (N) of a given plasmid within a population of cells is usually maintained within a narrow Gaussian distribution within a given host and under defined growth conditions. This is required, since plasmids must co-exist stably within their hosts and minimize metabolic load upon the cell. Specifically, over-accumulation of plasmid copies within a cell can slow cell growth and eventually cause cell death. In contrast, a plasmid replication rate that is too slow can lead to plasmid-free cells, since plasmid-free cells often grow faster and can outnumber plasmid-carrying cells in the population. Means for regulating plasmid copy number are the result of an autoregulatory control mechanism, wherein the plasmid DNA concentration itself determines the rate at which new plasmid copies are generated. In general, the initiation of plasmid replication may be controlled by regulating the amount of available primer for the initiation of DNA replication, regulating the amount of essential replication proteins, or regulating the function of essential replication proteins. Several recent reviews discuss details concerning plasmid copy number control (see, for example, Helinski, D. R., et al. Replication control and other stable maintenance mechanisms of plasmids. In Escherichia coli and Salmonella, Vol. 2. Neidhardt, F. C., et al. Eds. American Society of Microbiology: Washington, D.C., pp. 2295–2324 (1996); Chattoraj, D. K. and Schneider, T. D., Prog Nucleic Acid Res Mol Biol, 57:145–186 (1997); del Solar, G. & Espinosa, M., Mol Microbiol, 37(3): 492–500 (2000); and Chattoraj, D. K., Mol Microbiol, 37(3):467–476 (2000)).
A second consideration when selecting a particular expression vector is the host range of the vector itself. Specifically, host range refers to the types of microbes in which a plasmid will replicate. One may develop a specific vector for each microbial species of interest; or, one may take advantage of available broad host range replicons that have the ability to be maintained in a wide range of microbes that are unrelated. These broad host range plasmids typically encode all of their own proteins required for replication and which function in multiple hosts. Thus, these plasmids are not dependent on their host cell. In contrast, narrow host range replicons may lack replication or segregation proficiencies (as compared to an inability to be introduced into or express genetic markers in a distantly related host), which result in their replication only in closely related species (Schmidhauser, T. J. and D. R. Helinski., J. Bacteriol., 164:446–455 (1985)).
Most broad host range plasmids are classified on the basis of their intrinsic properties, according to their “incompatibility groups”. This classification reflects the similarities in sequence, function, and the nature of the replicon (as replicons of the same type are unable to co-exist in a cell, while replicons from different incompatibility groups (e.g., “Inc” groups) may exist simultaneously in a single cell). Natural plasmid isolates of Gram-negative bacteria that belong to Inc groups C, N, P, Q, and W display replication and maintenance proficiency in a diversity of bacterial species.
The pBBR1 plasmid is a 2.6 kB broad host range plasmid isolated from the Gram-negative bacterium Bordetella bronchiseptica S87 (Antoine, R. and C. Locht, Mol. Microbiol., 6(13):1785–1799 (1992); FR 2,690,459). Many derivatives of pBBR1 have been constructed to add various multiple cloning sites (Kovach et al., Biotechniques, 16: 800–802 (1994)), antibiotic resistance markers (Kovach et al., Gene, 166: 175–176 (1995)), reporter genes (Ramos et al., J Biotechnol, 97: 243–252 (2002)), and regulated promoters (Lefebre and Valvano, Appl Environ Microbiol, 68: 5956–5964 (2002); Sukchawalit et al., FEMS Microbiol Lett, 181: 217–223 (1999)). These pBBR1-based plasmid derivatives have been used in a variety of applications including: 1.) development of a genetic system for bacteria (Coppi et al., Appl Environ Microbiol, 67: 3180–3187 (2001); Su et al., Microbiology, 147: 581–589 (2001)); 2.) synthesis of novel polyhydroxy alkanoates (Ewering et al., Microbiology, 148: 1397–1406 (2002)); 3.) production of biocatalysts for biotransformation (Overhage et al., Appl Environ Microbiol, 68: 4315–4321 (2002)); and 4.) over-expression of a protective antigen to enhance vaccine efficacy (Vemulapalli et al., Infect Immun, 68: 3286–3289 (2000)).
One particular derivative of pBBR1 having utility as an expression/cloning vector with very broad host range maintenance is the commercially available pBHR1 (MoBiTec; Göttingen, Germany; GenBank® Y14439). Like pBBR1, pBHR1 does not belong to any of the common broad host range incompatibility groups and possesses a relatively high copy number. Both pBBR1 and pBHR1 plasmids possess two critical open reading frames (ORFs)—the first, known as rep, is involved in replication of the plasmid; and, the second ORF is known as mob. The mob gene, involved in mobilization, has been extensively characterized for this family of plasmids by Szpirer et al. (Molecular Microb. 37(6): 1283–1292 (2000); J. Bacteriol. 183(6): 2101–2110 (2001)). Plasmid pBHR1 also additionally has two selectable markers (i.e., kanamycin and chloramphenicol), while maintaining a relatively small size of only 5300 bp. These properties render pBHR1 an extremely useful cloning vector suitable for a wide range of Gram-negative bacteria.
One variation that would increase the utility of pBBR1 and plasmid derivatives within the pBBR1 family would be a means to increase the copy number of the plasmid. Specifically, it would be desirable to create a suite of mutants having altered plasmid copy number, since this would enable one to readily assess the relationship between gene copy number and gene expression. In general, increased plasmid copy number per cell can substantially increase the overall yield of proteins (i.e., titer) that are expressed by the plasmid within the host cell. Plasmid mutants having a phenotype of altered copy number are generated by random mutagenesis followed by screening to obtain mutants with the desired phenotype. Although the technique of generating these mutants is well understood by an artisan skilled in molecular biology, the utility and need for development of pBBR1-based plasmids having altered plasmid copy number has not previously been recognized.
The problem to be solved therefore is to develop a broad host range expression plasmid having the ability to: 1.) co-exist with a variety of other broad host range plasmids; and 2.) replicate within a given host under defined growth conditions, such that the plasmid copy number is altered relative to the native pBBR1-based plasmid.
The present problem has been solved by providing a suite of isolated plasmids derived from pBHR1 comprised of mutant replication control regions conveying a phenotype of increased plasmid copy number. The broad host range of the plasmids, and their compatibility with other known broad host range vectors, makes the plasmids of the present invention particularly attractive for plasmid-based protein expression within a variety of bacteria.